Clostridial toxin disease therapy

ABSTRACT

Treating humans and animals intoxicated with a bacterial toxin by administration of antitoxin. Avian antitoxin in an aqueous solution in therapeutic amount that is orally administrable.

[0001] This application is a continuation-in-part of application Ser.No. 429,791, filed Oct. 31, 1989.

FIELD OF THE INVENTION

[0002] The present invention relates to Clostridial antitoxin therapyfor humans and animals.

BACKGROUND OF THE INVENTION

[0003] Several species of Clostridia bacteria produce toxins ofsignificance to human and animal health [C. L. Hatheway, Clin.Microbiol. Rev., 3:66-98 (1990)]. The effects of these toxins range fromdiarrheal diseases that can cause destruction of the colon, to paralyticeffects that can cause death. Particularly at risk for developingClostridial diseases are neonates and humans and animals in poor health(e.g., those suffering from diseases associated with old age orimmunodeficiency diseases).

[0004] The bacterium Clostridium botulinum produces the most poisonousbiological toxin known. The lethal human dose is a mere 10⁻⁹ mg/kgbodyweight for toxin in the bloodstream. Botulinal toxin blocks nervetransmission to the muscles, resulting in flaccid paralysis. When thetoxin reaches airway and respiratory muscles, it results in respiratoryfailure that can cause death. [S. Arnon, J. Infectious Diseases,154:201-206 (1986).]

[0005]C. botulinum is a spore-forming anaerobe whose habitat is soil andthe mud of lakes and ponds. The spores are carried by dust and are foundon vegetables taken from the soil, on fresh fruits, and on agriculturalproducts such as honey. Under conditions favorable to the organism, thespores germinate to vegetative cells which produce toxin. [S. Arnon, AnnRev. Med., 31:541 (1980).]

[0006] Botulism disease may be grouped into three types, based on themethod of introduction of toxin into the bloodstream. Food-bornebotulism results from ingesting improperly preserved and inadequatelyheated food that contains botulinal toxin. There were 355 cases offood-borne botulism in the United States between 1976 and 1984. [K. L.MacDonald et al., Am. J. Epidemiology, 124:794 (1986).] The death ratedue to botulinal toxin is 12% and can be higher in particular riskgroups. [C. O. Tacket et al., Am. J. Med., 76:794 (1984).] Wound-inducedbotulism results from C. botulinum penetrating traumatized tissue andproducing toxin that is absorbed into the bloodstream. Since 1950,thirty cases of wound botulism have been reported. [M. N. Swartz,Microbiology, 4th ed. (1990).] Infectious infant botulism results fromC. botulinum colonization of the infant intestine with production oftoxin and its absorption into the bloodstream. It is likely that thebacterium gains entry when spores are ingested and subsequentlygerminate. [S. Arnon, J. Infectious Diseases, 154:201-206 (1986).] Therehave been 500 cases reported since it was first recognized in 1976. [M.N. Swartz, supra.]

[0007] Infant botulism strikes infants who are three weeks to elevenmonths old (greater than 90% of the cases are infants less than sixmonths). [S. Arnon, J. Infectious Diseases, 154:201-206 (1986).] It isbelieved that infants are susceptible, due, in large part, to theabsence of the full adult complement of intestinal microflora. Thebenign microflora present in the adult intestine provide an acidicenvironment that is not favorable to colonization by C. botulinum.Infants begin life with a sterile intestine which is gradually colonizedby microflora. Because of the limited microflora present in earlyinfancy, the intestine is not as acidic, allowing for C. botulinum sporegermination, growth, and toxin production In this regard, some adultswho have undergone antibiotic therapy which alters intestinal microflorabecome more susceptible to botulism.

[0008] An additional factor accounting for infant susceptibility toinfectious botulism is the immaturity of the infant immune system. Themature immune system is sensitized to bacterial antigens and producesprotective antibodies. Secretory IgA produced in the adult intestine hasthe ability to agglutinate vegetative cells of C. botulinum. [S. Arnon,J. Infectious Diseases, 154:201-206 (1986).] Secretory IgA may also actby preventing intestinal bacteria and their products from crossing thecells of the intestine. [S. Arnon, Epidemiologic Reviews, 3:45-66(1981).] The infant immune system is not primed to do this.

[0009] Clinical symptoms of infant botulism range from mild paralysis,to moderate and severe paralysis requiring hospitalization, to fulminantparalysis, leading to sudden death. [S. Arnon et al., EpidemiologicReviews, 3:45-66 (1981).]

[0010] The chief therapy for severe infant botulism is ventilatoryassistance using a mechanical respirator and concurrent elimination oftoxin and bacteria using cathartics, enemas, and gastric lavage. Therewere 68 hospitalizations in California for infant botulism in a singleyear with a total cost of over $4 million for treatment. [T. L.Frankovich & S. Arnon, West. J. Med., 154:103 (1991).]

[0011] Different types of Clostridium each produce antigenicallydistinct toxin designated by the letters A-G. Nearly all cases of infantbotulism have been caused by bacteria producing either type A or type Btoxin. (Exceptionally, one New Mexico case was caused by Clostridiumproducing type F toxin and another by Clostridium producing a typeB-type F hybrid.) [S. Arnon, Epidemiologic Reviews, 3:45-66 (1981).]Type C toxin affects waterfowl, type D toxin affects cattle, and type Etoxin affects both humans and birds.

[0012] A trivalent antitoxin derived from horse plasma is commerciallyavailable from Connaught Industries Ltd. as a therapy for toxin types A,B, and E. However the antitoxin has several disadvantages. First,extremely large dosages must be injected intravenously and/orintramuscularly. Second, the antitoxin has serious side effects such asacute anaphylaxis which can lead to death, and serum sickness. Finally,the efficacy of the antitoxin is uncertain and the treatment is costly.[T. O. Tacket et al., Am. J. Med., 76:794-98 (1984).]

[0013] A heptavalent equine botulinal antitoxin which uses only theF(ab′)2 portion of the antibody molecule has been tested by the UnitedStates Military. [M. Balady, USAMRDC Newsletter, p. 6 (1991).] This wasraised against impure toxoids in those large animals and is not a hightiter preparation.

[0014] A pentavalent human antitoxin has been collected from immunizedhuman subjects for use as a treatment for infant botulism. The supply ofthis antitoxin is limited and cannot be expected to meet the needs ofall individuals stricken with botulism disease. In addition, collectionof human sera must involve screening out HIV and other potentiallyserious human pathogens. [P. J. Schwarz & S. S. Arnon, Western J. Med.,156:197-98 (1992).]

[0015] Infant botulism has been implicated as the cause of mortality insome cases of Sudden Infant Death Syndrome (SIDS, also known as cribdeath). SIDS is officially recognized as infant death that is sudden andunexpected and that remained unexplained despite complete post-mortemexamination. The link of SIDS to infant botulism came when fecal orblood specimens taken at autopsy from SIDS infants were found to containC. botulium organisms and/or toxin in 3-4% of cases analyzed. [D. R.Peterson et al., Reviews of Infect. Dis., 1:630-34 (1979).] In contrast,only 1 of 160 healthy infants (0.6%) had C. botulinum organisms in thefeces and no botulinal toxin. [S. Arnon et al., The Lancet, pp. 1273-77,Jun. 17, 1978.]

[0016] In developed countries, SIDS is the number one cause of death inchildren between one month and one year old. [S. Arnon et al., TheLancet, pp. 1273-77, Jun. 17, 1978.] More children die from SIDS in thefirst year than from any other single cause of death in the firstfourteen years of life. In the United States, there are 8,000-10,000SIDS victims annually. Id.

[0017] What is needed is an effective therapy against infant botulismthat is free of dangerous side effects, is available in large supply ata reasonable price, and can be safely and gently delivered so thatprophylactic application to infants is feasible.

SUMMARY OF THE INVENTION

[0018] The present invention contemplates treating humans and animalsintoxicated with a bacterial toxin by oral administration of antitoxinraised against the toxin. In one embodiment, the present inventioncontemplates a method of treatment comprising: a) providing, i) avianantitoxin in an aqueous solution in therapeutic amount that is orallyadministrable, and ii) at least one intoxicated subject; and b) orallyadministering the avian antitoxin to subject.

[0019] The present invention further contemplates a method ofprophylactic treatment comprising: a) providing i) avian antitoxin in anaqueous solution in therapeutic amount that is orally administrable, andii) at least one infant subject; and b) orally administering the avianantitoxin to subject.

[0020] The invention is particularly useful where antitoxin comprisesClostridial antitoxin such as Clostridial botulinus antitoxin. In oneembodiment, the present invention contemplates that the aqueous solutioncomprises a nutritional formula, such as infant formula.

[0021] As a composition, avian clostridial antitoxin, such as botulinusantitoxin, is contemplated wherein it is in an aqueous solution intherapeutic amounts that is orally administrable. In a preferredembodiment, the aqueous solution is a nutritional formula such as infantformula.

[0022] The present invention also contemplates a method of treatment ofenteric bacterial infections comprising: a) providing i) avian antitoxinin an aqueous solution in therapeutic amount that is orallyadministrable, and ii) at least one infected subject; and b) orallyadministering avian antitoxin to the subject. Such bacterial infectionsmay be infections of Clostridium botulinus, Clostridium butyicum,Clostridium difficile, Clostridium perfringens, and Clostridiumsordelli. The antitoxin may comprise botulinus antitoxin and the aqueoussolution may comprises a nutritional formula, such as infant formula.

[0023] The present invention also contemplates diagnostic uses for theantitoxins. In one embodiment, the present invention contemplates amethod for detecting Clostridial toxin in biological tissues comprising:a) providing, i) a biological tissue, ii) an avian antitoxin raisedagainst the Clostridial toxin, and iii) a reporter antibody substancewith binding specificity for the antitoxin; b) adding the antitoxin tobiological tissue so that the antitoxin binds to the Clostridial toxinin the biological tissue; c) washing the unbound antitoxin from thebiological tissue; d) adding the reporter antibody substance to thebiological tissue so that the reporter antibody substance binds to boundantitoxin; e) washing the unbound reporter antibody substance from thebiological tissue; and f) detecting the reporter antibody substancebound to the antitoxin bound to the Clostridial toxin so that theClostridial toxin is detected. In a preferred embodiment, theClostridial toxin is botulinal toxin.

DESCRIPTION OF THE DRAWINGS

[0024]FIG. 1 shows the reactivity of antibotulinum IgY by Western blot.

[0025]FIG. 2 shows the IgY antibody titer to botulinum type A toxoid ofeggs by ELISA.

DESCRIPTION OF THE INVENTION

[0026] The present invention contemplates treating humans and animalsintoxicated with a bacterial toxin by oral administration of antitoxinraised against the toxin. The individual steps of are describedseparately below.

[0027] I. Obtaining Antibodies in Non-Mammals

[0028] A preferred embodiment of the method of the present invention forobtaining antibodies involves immunization. However, it is alsocontemplated that antibodies could be obtained from non-mammals withoutimmunization. In the case where no immunization is contemplated, thepresent invention may use non-mammals with preexisting antibodies totoxins as well as non-mammals that have antibodies to whole organisms byvirtue of reactions with the administered antigen. An example of thelatter involves immunization with synthetic peptides or recombinantproteins sharing epitopes with whole organism components.

[0029] In a preferred embodiment, the method of the present inventioncontemplates immunizing non-mammals with bacterial toxin(s). It is notintended that the present invention be limited to any particular toxin.In one embodiment, toxin from all Clostridial bacteria sources (seeTable 1) are contemplated as immunogens. Examples of these toxins are C.butyicum neruraminidase toxin, C. difficile toxins A and B, C.perfringens toxins α, β, E, and ι, and C. sordelli toxins HT and LT. Ina preferred embodiment, toxins A, B, C, D, E, F, and G from C. botulinumare contemplated as immunogens. TABLE 1 Clostridial Toxins and DiseasesTOXINS DISEASE ASSOCIATION C. botulinum A, B, C, D, E, Infant botulism,food F, G poisoning C. butyicum neuraminidase botulism, enterocolitis?C. difficile A, B pseudomembranous colitis, antibiotic- associateddiarrhea, enterocolitis? C. perfringens alpha, beta, gas gangrene, foodepsilon, iotatoxins poisoning C. sordelli HT, LT diarrhea, gas gangrene

[0030] When immunization is used, the preferred non-mammal is from theclass Aves. All birds are contemplated (e.g., duck, ostrich, emu,turkey, etc.). A preferred bird is a chicken. Importantly, chickenantibody does not fix mammalian complement. (See H. N. Benson et al., J.Immunol., 87:610 (1961).) Thus, chicken antibody will normally not causea complement-dependent reaction. [A. A. Benedict and K. Yamaqa,Comparative Immunology, (J. J. Marchaloni, ed.), Ch. 13,“Immunoglobulins and Antibody Production in Avian Species,” pp. 335-375,Blackwell, Oxford (1966).] Thus, the preferred antitoxins of the presentinvention will not exhibit complement-related side effects observed withantitoxins known presently.

[0031] When birds are used, it is contemplated that the antibody will beobtained from either the bird serum or the egg. A preferred embodimentinvolves collection of the antibody from the egg. Laying hens exportimmunoglobulin to the egg yolk (“IgY”) in concentrations equal to orexceeding that found in serum. (See R. Patterson et al., J. Immunol.,89:272 (1962).) [S. B. Carroll and B. D. Stollar, J. Biol. Chem., 258:24(1983).] In addition, the large volume of egg yolk produced vastlyexceeds the volume of serum that can be safely obtained from the birdover any given time period. Finally, the antibody from eggs is purer andmore homogeneous; there is far less non-immunoglobulin protein (ascompared to serum) and only one class of immunoglobulin is transportedto the yolk.

[0032] When considering immunization with toxins, one may considermodification of the toxins to reduce its toxicity. In this regard, it isnot intended that the present invention be limited by immunization withmodified toxin. Unmodified (“native”) toxin is also contemplated as animmunogen.

[0033] It is also not intended that the present invention be limited bythe type of modification—if modification is used. The present inventioncontemplates all types of toxin modification, including chemical andheat treatment of the toxin. The preferred modification, however, isformaldehyde treatment.

[0034] It is not intended that the present invention be limited to aparticular mode of immunization; the present invention contemplates allmodes of immunization, including subcutaneous, intramuscular,intraperitoneal, and intravascular injection.

[0035] The present invention further contemplates immunization with orwithout adjuvant. (Adjuvant is defined as a substance known to increasethe immune response to other antigens when administered with otherantigens.) If adjuvant is used, it is not intended that the presentinvention be limited to any particular type of adjuvant—or that the sameadjuvant, once used, be used all the time. While the present inventioncontemplates all types of adjuvant, whether used separately or incombinations, the preferred use of adjuvant is the use of CompleteFreund's Adjuvant followed sometime later with Incomplete Freund'sAdjuvant.

[0036] When immunization is used, the present invention contemplates awide variety of immunization schedules. In one embodiment, a chicken isadministered toxin(s) on day zero and subsequently receives toxin(s) inintervals thereafter. It is not intended that the present invention belimited by the particular intervals or doses. Similarly, it is notintended that the present invention be limited to any particularschedule for collecting antibody. The preferred collection time issometime after day 100.

[0037] Where birds are used and collection of antibody is performed bycollecting eggs, the eggs may be stored prior to processing forantibody. It is preferred that storage of the eggs be performed at 4° C.for less than one year.

[0038] It is contemplated that chicken antibody produced in this mannercan be buffer-extracted and used analytically. While unpurified, thispreparation can serve as a reference for activity of the antibody priorto further manipulations (e.g. immunoaffinity purification).

[0039] II. Increasing the Effectiveness of Antibodies

[0040] When purification is used, the present invention contemplatespurifying to increase the effectiveness of both non-mammalian antitoxinsand mammalian antitoxins. Specifically, the present inventioncontemplates increasing the percent of toxin-reactive immunoglobulin.The preferred purification approach for avian antibody is PolyethyleneGlycol (PEG) separation.

[0041] A. PEG Purification

[0042] The present invention contemplates that avian antibody beinitially purified using simple, inexpensive procedures. In oneembodiment, chicken antibody from eggs is purified by extraction andprecipitation with polyethylene glycol (PEG). PEG purification exploitsthe differential solubility of lipids (which are abundant in egg yolks)and yolk proteins in high concentrations of polyethylene glycol 8000.[Polson et al., Immunol. Comm., 9:495 (1980).1 The technique is rapid,simple, and relatively inexpensive and yields an immunoglobulin fractionthat is significantly purer in terms of contaminating non-immunoglobulinproteins than the comparable ammonium sulfate fractions of mammaliansera and horse antibodies. Indeed, PEG-purified antibody is sufficientlypure that the present invention contemplates the use of PEG-purifiedantitoxins in the passive immunization of intoxicated humans andanimals.

[0043] III. Treatment

[0044] The present invention contemplates antitoxin therapy for humansand animals intoxicated by bacterial toxins. A preferred method oftreatment is by oral administration of antitoxin.

[0045] A. Dosage of Antitoxin It was noted by way of background that abalance must be struck when administering currently available antitoxin;sufficient antitoxin must be administered to neutralize the toxin, butnot so much antitoxin as to increase the risk of untoward side effects.These side effects are caused by i) patient sensitivity to horseproteins, ii) anaphylactic or immunogenic properties ofnon-immunoglobulin proteins, iii) the complement fixing properties ofmammalian antibodies, and/or iv) the overall burden of foreign proteinadministered. It is extremely difficult to strike this balance when, asnoted above, the degree of intoxication (and hence the level ofantitoxin therapy needed) can only be approximated.

[0046] The present invention contemplates significantly reducing sideeffects so that this balance is more easily achieved. Treatmentaccording to the present invention contemplates reducing side effects byusing PEG-purified antitoxin from birds.

[0047] In one embodiment, the treatment of the present inventioncontemplates the use of PEG-purified antitoxin from birds. The use ofyolk-derived, PEG-purified antibody as antitoxin allows for theadministration of 1) non(mammalian)-complement-fixing, avian antibody,2) a less heterogeneous mixture of non-immunoglobulin proteins, and 3)less total protein to deliver the equivalent weight of active antibodypresent in currently available antitoxins. The non-mammalian source ofthe antitoxin makes it useful for treating patients who are sensitive tohorse or other mammalian serums.

[0048] B. Delivery of Antitoxin

[0049] The present invention contemplates a method for antitoxintreatment of bacterial intoxication in which delivery of antitoxin in anaqueous solution. The solution has sufficient ionic strength tosolubilize antibody protein yet is made palatable for oraladministration. In one embodiment the delivery solution is an aqueoussolution. In another embodiment the delivery solution is a nutritionalformula. Preferably, the delivery solution is infant formula.

[0050] The invention contemplates a method of treatment which can beadministered for treatment of acute intoxication. In one embodiment,antitoxin is administered orally, in a delivery solution, in therapeuticdosage, to a subject intoxicated by the bacterial toxin which served asimmunogen for the antitoxin.

[0051] The invention also contemplates a method of treatment which canbe administered prophylactically. In one embodiment, antitoxin isadministered orally, in a delivery solution, in therapeutic dosage, to asubject, to prevent intoxication of the subject by the bacterial toxinwhich served as immunogen for the antitoxin. In a preferred embodimentthe subject is an infant. In another embodiment, antibody raised againstwhole bacterial organism is administered orally to a subject, in adelivery solution, in therapeutic dosage.

[0052] IV. Detection of Toxin

[0053] The invention contemplates detecting bacterial toxin inbiological tissues, by a method that utilizes antitoxin raised againstthe toxin and a reporter substance. The reporter substance comprises anantibody with binding specificity for the antitoxin attached to amolecule which is used to identify the presence of the reportersubstance. The biological tissue is first exposed to antitoxin whichbinds to toxin and is then washed free of substantially all unboundantitoxin. The biological tissue is next exposed to the reportersubstance, which binds to antitoxin and is then washed free ofsubstantially all unbound reporter substance. Identification of thereporter substance in the biological tissue indicates the presence ofthe bacterial toxin.

EXPERIMENTAL

[0054] The following examples serve to illustrate certain preferredembodiments and aspects of the present invention and are not to beconstrued as limiting the scope thereof.

[0055] In the disclosure which follows, the following abbreviationsapply: ° C. (degrees Centigrade); rpm (revolutions per minute);BBS-Tween (borate buffered saline containing Tween); BSA (bovine serumalbumin); ELISA (enzyme-linked immunosorbent assay); CFA (completeFreund's adjuvant); IFA (incomplete Freund's adjuvant); IgG(immunoglobulin G); IgY (immunoglobulin Y); H₂O (water); HCl(hydrochloric acid); LD₁₀₀ (lethal dose for 100% of experimentalanimals); kD (kilodaltons); gm (grams); μg (micrograms); mg(milligrams); ng (nanograms); μl (microliters); ml (milliliters); mm(millimeters); nm (nanometers); μm (micrometer); M (molar); mM(millimolar); MgCl₂ (magnesium chloride); NaCl (sodium chloride); Na₂CO₃(sodium carbonate); OD₂₈₀ (optical density of 280 nm); PAGE(polyacrylamide gel electrophoresis); PBS (phosphate buffered saline(150 mM NaCl, 10 mM sodium phosphate buffer, pH 7.2)); PEG (polyethyleneglycol); SDS (sodium dodecylsulfate); Tris(tris(hydroxymethyl)aminomethane);w/v (weight to volume); v/v (volume tovolume); Amresco (Solon, Ohio); BBL (BBL, Cockeysville, Md.); BioRad(BioRad, Richmond, Calif.); Fisher Biotech (Fisher Biotech, Springfield,N.J.); GIBCO (GIBCO, Grand Island, N.Y.)); Millipore (Millipore Corp.,Marlborough, Mass.); (Ross Laboratories, Columbus, Ohio); Sigma (SigmaChemical Co., St. Louis, Mo.);

Example 1 Production of High-Titer Antibodies to Clostridium DifficileOrganisms in a Hen

[0056] Antibodies to certain pathogenic organisms have been shown to beeffective in treating diseases caused by those organisms. It has notbeen shown whether antibodies can be raised, against the Clostridiumpathogen, which would be effective in treating infection by thisorganism. Accordingly, C. difficile was tested as immunogen forproduction of hen antibodies.

[0057] To determine the best course for raising high-titer eggantibodies against whole C. difficile organisms, different immunizingstrains and different immunizing concentrations were examined. Theexample involved (a) preparation of the bacterial immunogen, (b)immunization, (c) purification of anti-bacterial chicken antibodies, and(d) detection of anti-bacterial antibodies in the purified IgYpreparations.

[0058] (a) Preparation of Bacterial Immunogen. C. difficile strains43594 (serogroup A) and 43596 (serogroup C) were originally obtainedfrom the American Type Culture Collection, Rockville, Md. These twostrains were selected because they represent two of the mostcommonly-occurring serogroups isolated from patients withantibiotic-associated pseudomembranous colitis. [Delmee et al., J. Clin.Microbial., 28(10):2210 (1990).] Additionally, both of these strainshave been previously characterized with respect to their virulence inthe Syrian Hamster Model for C. difficile infection. [Delmee et al., J.Med Microbial., 33:85 (1990).]

[0059] The bacterial strains were separately cultured on brain heartinfusion agar for 48 hours at 37° C. in a Gas Pack 100 Jar (BBL,Cockeysville, Md.) equipped with a Gas Pack Plus anaerobic envelope(BBL). Forty-eight hour cultures were used because they produce bettergrowth and the organisms have been found to be more cross-reactive withrespect to their surface antigen presentation. The greater the degree ofcross-reactivity of our IgY preparations, the better the probability ofa broad range of activity against different strains/serogroups. (Toma etal., J. Clin. Microbiol., 26(3):426 (1988).]

[0060] The resulting organisms were removed from the agar surface usinga sterile dacron-tip swab, and were suspended in a solution containing0.4% formaldehyde in PBS, pH 7.2. This concentration of formaldehyde hasbeen reported as producing good results for the purpose of preparingwhole-organism immunogen suspensions for the generation of polyclonalanti-C. difficile antisera in rabbits. [Delmee et al., J. Clin.Microbiol., 21(3):323 (1985); Davies et al., Microbial Path., 9:141(1990).] In this manner, two separate bacterial suspensions wereprepared, one for each strain. The two suspensions were then incubatedat 4° C. for 1 hour Following this period of formalin-treatment, thesuspensions were centrifuged at 4,200×g for 20 min., and the resultingpellets were washed twice in normal saline. The washed pellets, whichcontained formalin-treated whole organisms, were resuspended in freshnormal saline such that the visual turbidity of each suspensioncorresponded to a #7 McFarland standard. [S. M. Finegold et al., Baileyand Scott's Diagnostic Microbiology, pp. 488-489, The C. V. Mosby Co.,(1978).] Each of the two #7 suspensions was then split into two separatevolumes. One volume of each suspension was volumetrically adjusted, bythe addition of saline, to correspond to the visual turbidity of a #1McFarland standard. [S. M. Finegold et al., Bailey and Scott'sDiagnostic Microbiology, pp. 488-489, The C. V. Mosby Co., (1978).] The#1 suspensions contained approximately 3×10⁸ organisms/ml, and the #7suspensions contained approximately 2×10⁹ organisms/ml. [S. M. Finegoldet al., Bailey and Scott's Diagnostic Microbiology, pp. 488-489, The C.V. Mosby Co., (1978).] The four resulting concentration-adjustedsuspensions of formalin-treated C. difficile organisms were consideredto be “bacterial immunogen suspensions.” These suspensions were usedimmediately after preparation for the initial immunization (see section(b)).

[0061] The formalin-treatment procedure did not result in 100%non-viable bacteria in the immunogen suspensions. In order to increasethis, the formalin concentration and length of treatment were bothincreased for subsequent immunogen preparations, as described below inTable 3. (Although viability was decreased with the stronger formalintreatment, 100% inviability of the bacterial immunogen suspensions wasnot reached.) Also, in subsequent immunogen preparations, the formalinsolutions were prepared in normal saline instead of PBS. At day 49, theday of the fifth immunization, the excess volumes of the four previousbacterial immunogen suspensions were stored frozen at −70° C. for useduring all subsequent immunizations.

[0062] (b) Immunization. For the initial immunization, 1.0 ml volumes ofeach of the four bacterial immunogen suspensions described above wereseparately emulsified in 1.2 ml volumes of CFA (GIBCO, Grand Island,N.Y.). For each of the four emulsified immunogen suspensions, twofour-month old White Leghorn hens (pre-laying) were immunized. (It isnot necessary to use pre-laying hens; actively-laying hens can also beutilized.) Each hen received a total volume of approximately 1.0 ml of asingle emulsified immunogen suspension via four injections (twosubcutaneous and two intramuscular) of approximately 250 μl per site. Inthis manner, a total of four different immunization combinations, usingtwo hens per combination, were initiated for the purpose of evaluatingboth the effect of immunizing concentration on egg yolk antibody (IgY)production, and interstrain cross-reactivity of IgY raised againstheterologous strains. The four immunization groups are summarized inTable 2. TABLE 2 Immunization Groups GROUP IMMUNIZING APPROXIMATEDESIGNATION STRAIN IMMUNIZING DOSE CD 43594, #1 C diff. 43594 1.5 × 10⁸org./hen CD 43594, #7 ″ 1.0 × 10⁹ org./hen CD 43596, #1 C. diff. 435961.5 × 10⁸ org./hen CD 43596, #7 ″ 1.0 × 10⁹ org./hen

[0063] The time point for the first series of immunizations wasdesignated as “day zero.” All subsequent immunizations were performed asdescribed above except that the bacterial immunogen suspensions wereemulsified using IFA (GIBCO) instead of CFA, and for the later timepoint immunization, the stored frozen suspensions were used instead offreshly-prepared suspensions. The immunization schedule used is listedin Table 3. TABLE 3 Immunization Schedule DAY OF FORMALIN- IMMUNOGENPREPARATION IMMUNIZATION TREATMENT USED 0 1%, 1 hr. freshly-prepared 141%, overnight ″ 21 1%, overnight ″ 35 1%, 48 hrs. ″ 49 1%, 72 hrs. ″ 70″ stored frozen 85 ″ ″ 105 ″ ″

[0064] (c) Purification of Anti-Bacterial Chicken Antibodies. Groups offour eggs were collected per immunization group between days 80 and 84post-initial immunization, and chicken immunoglobulin (IgY) wasextracted according to a modification of the procedure of A. Polson etal. [Immunol. Comm., 9:495 (1980)]. A gentle stream of distilled waterfrom a squirt bottle was used to separate the yolks from the whites, andthe yolks were broken by dropping them through a funnel into a graduatedcylinder. The four individual yolks were pooled for each group. Thepooled, broken yolks were blended with 4 volumes of egg extractionbuffer to improve antibody yield (egg extraction buffer is 0.01 M sodiumphosphate, 0.1 M NaCl, pH 7.5, containing 0.005% Thimerosal), and PEG8000 (Amresco) was added to a concentration of 3.5%. When all the PEGdissolved, the protein precipitates that formed were pelleted bycentrifugation at 13,000×g for 10 minutes. The supernatants weredecanted and filtered through cheesecloth to remove the lipid layer, andthe PEG was added to the supernatants to a final concentration of 12%(the supernatants were assumed to contain 3.5% PEG). After a secondcentrifugation, the supernatants were discarded and the pellets werecentrifuged a final time to extrude the remaining PEG. These crude IgYpellets were then dissolved in the original yolk volume of eggextraction buffer and stored at 4° C. As an additional control, apreimmune IgY solution was prepared as described above, using eggscollected from unimmunized hens.

[0065] (d) Detection of Anti-Bacterial Antibodies in the Purified IgYPreparations. In order to evaluate the relative levels of specificanti-C. difficile activity in the IgY preparations described above, amodified version of the whole-organism ELISA procedure of N. V. Padhyeet al. [J. Clin. Microbiol. 29:99-103] was used. Frozen organisms ofboth C. difficile strains described above were thawed and diluted to aconcentration of approximately 1×10⁷ org./ml using PBS, pH 7.2. In thisway, two separate coating suspensions were prepared, one for eachimmunizing strain. Into the wells of 96-well microtiter plates (Falcon,Pro-Bind Assay Plates) were placed 100 μl volumes of the coatingsuspensions. In this manner, each plate well received a total ofapproximately 1×10⁶ organisms of one strain or the other. The plateswere then incubated at 4° C. overnight. The next morning, the coatingsuspensions were decanted, and all wells were washed three times usingPBS. In order to block non-specific binding sites, 100 μl of 0.5% BSA(Sigma, St. Louis, Mo.) in PBS was then added to each well, and theplates were incubated for 2 hours at room temperature. The blockingsolution was decanted, and 100 μl volumes of the IgY preparationsdescribed above were initially diluted 1:500 with a solution of 0.1% BSAin PBS, and then serially diluted in 1:5 steps. The following dilutionswere placed in the wells: 1:500, 1:2,500, 1:62,5000, 1:312,500, and1:1,562,500. The plates were again incubated for 2 hours at roomtemperature. Following this incubation, the IgY-containing solutionswere decanted, and the wells were washed three times using BBS-tween(0.1 M boric acid, 0.025 M sodium borate, 1.0 M NaCl, 0.1% tween-20),followed by two washes using PBS-tween (0.1% tween-20), and finally, twowashes using PBS only. To each well, 100 μl of a 1:750 dilution ofrabbit anti-chicken IgG (whole-molecule)-alkaline phosphatase conjugate(Sigma, St. Louis, Mo.) (diluted in 0.1% BSA in PBS) was added. Theplates were again incubated for 2 hours at room temperature. Theconjugate solutions were decanted and the plates were washed asdescribed above, substituting 50 mM Na₂Co₃, pH 9.5 for the PBS in thefinal wash. The plates were developed by the addition of 100 μl of asolution containing 1 mg/ml para-nitro phenyl phosphate (Sigma, St.Louis, Mo.) dissolved in 50 mM Na₂CO₃, 10 mM MgCl₂₁ pH 9.5 to each well,and incubating the plates at room temperature in the dark for 45minutes. The absorbance of-each well was measured at 410 nm using aDynatech MR 700 plate reader. In this manner, each of the four IgYpreparations described above were tested for reactivity against both ofthe immunizing C. difficile strains, and strain-specific, as well ascross-reactive activity was determined. TABLE 4 Results of the Anti-C.Difficile Whole-Organism ELISA IgY DILUTION 43594-COATED 43596-COATEDPREPARATION IgY PREP WELLS WELLS Cd 43594, #1 1:500 1.746 1.801 1:2,5001.092 1.670 1:12,500 0.202 0.812 1:62,500 0.136 0.179 1:312,500 0.0120.080 1:1,562,500 0.002 0.020 CD 43594, #7 1:500 1.780 1.771 1:2,5001.025 1.078 1:12,500 0.188 0.382 1:62,500 0.052 0.132 1:312,500 0.0220.043 1:1,562,500 0.005 0.024 CD 43596, #1 1:500 1.526 1.790 1:2,5000.832 1.477 1:12,500 0.247 0.452 1:62,500 0.050 0.242 1:312,500 0.0100.067 1:1,562,500 0 0.036 CD 43596, #7 1:500 1.702 1.505 1:2,500 0.7060.866 1:12,500 0.250 0.282 1:62,500 0.039 0.078 1:312,500 0.002 0.0171:1,562,500 0 0.010 Preimmune IgY 1:500 0.142 0.309 1:2,500 0.032 0.0771:12,500 0.006 0.024 1:62,500 0.002 0.012 1:312,500 0.004 0.0101:1,562,500 0.002 0.014

[0066] Table 4 shows the results of the whole-organism ELISA. All fourIgY preparations demonstrated significant levels of activity, to adilution of 1:62,500 or greater against both of the immunizing organismstrains. Therefore, antibodies raised against one strain were highlycross-reactive with the other strain, and vice versa. The immunizingconcentration of organisms did not have a significant effect onorganism-specific IgY production, as both concentrations producedapproximately equivalent responses. Therefore, the lower immunizingconcentration of approximately 1.5×108 org./hen is the preferredimmunizing concentration of the two tested. The preimmune IgYpreparation appeared to possess relatively low levels of C.difficile-reactive activity to a dilution of 1:500, probably due toprior exposure of the animals to environmental Clostridia.

[0067] An initial whole-organism ELISA was performed using IgYpreparations made from single CD 43594, #1 and CD 43596, #1 eggscollected around day 50 (data not shown). Specific titers were found tobe S to 10-fold lower than those reported in Table 4. These resultsdemonstrate that it is possible to begin immunizing hens prior to thetime that they begin to lay eggs, and to obtain high titer specific IgYfrom the first eggs that are laid. In other words, it is not necessaryto wait for the hens to begin laying before the immunization schedule isstarted.

Example 2 Treatment of C. Difficile Infection with Anti-C. DifficileAntibody

[0068] In order to determine whether the immune IgY antibodies raisedagainst whole C. difficile organisms were capable of inhibiting theinfection of hamsters by C. difficile, hamsters infected by thesebacteria were utilized [Lyer et al., Infection and Immunity,59:2215-2218 (1991)]. This example involved: (a) determination of thelethal dose of C. difficile organisms; and (b) treatment of infectedanimals with immune antibody or control antibody in nutritionalsolution.

[0069] (a) Determination of the Lethal Dose of C. Difficile Organisms.Determination of the lethal dose of C. difficile organisms was carriedout according to the model described by D. M. Lyerly et al. [Infect. andImmun., 59:2215-2218 (1991)]. C. difficile strain ATCC 43596 (serogroupC, American Type Culture Collection) was plated on BHI agar and grownanaerobically (BBL Gas Pak 100 system) at 37° C. for 42 hours. Organismswere removed from the agar surface using a sterile dacron-tip swab andsuspended in sterile 0.9% NaCl to a density of 10⁸ organisms/ml.

[0070] In order to determine the lethal dose of C. difficile in thepresence of control antibody and nutritional formula, non-immune eggswere obtained from unimmunized hens and a 12% PEG preparation made asdescribed in Example 1(c). This preparation was redissolved in onefourth the original yolk volume of Ensure, vanilla flavor (RossLaboratories, Columbus, Ohio).

[0071] Starting on day one, groups of female Golden Syrian hamsters(Harland Sprague Dawley, Madison, Wis.), 8-9 weeks old and weighingapproximately 100 gm, were orally administered 1 ml of thepreimmune/Ensure formula at time zero, 2 hours, 6 hours, and 10 hours At1 hour, animals were orally administered 3.0 mg Clindamycin HCl (Sigma)in 1 ml of water. This drug predisposes hamsters to C. difficileinfection by altering the normal intestinal flora. On day two, theanimals were given 1 ml of the preimmune IgY/Ensure formula at timezero, 2 hours, 6 hours, and 10 hours. At 1 hour on day two, differentgroups of animals were inoculated orally with saline (control), or 10²,10⁴, 10⁶, or 10⁸ C. difficile organisms in 1 ml of saline. From days3-12, animals were given 1 ml of the preimmune IgY/Ensure formula threetimes daily and observed for the onset of diarrhea and death. Eachanimal was housed in an individual cage and was offered food and waterad libitum.

[0072] Administration of 10⁶-10⁸ organisms resulted in death in 3-4 dayswhile the lower doses of 10²-10⁴ organisms caused death in 5 days. Cecalswabs taken from dead animals indicated the presence of C. difficile.Given the effectiveness of the 10² dose, this number of organisms waschosen for the following experiment to see if hyperimmune anti-C.difficile antibody could block infection.

[0073] (b) Treatment of Infected Animals with Immune Antibody or ControlAntibody in Nutritional Formula. The experiment in (a) was repeatedusing three groups of seven hamsters each. Group A received noclindamycin or C. difficile and was the survival control. Group Breceived clindamycin, 10² C. difficile organisms and preimmune IgY onthe same schedule as the animals in (a) above. Group C receivedclindamycin, 102 C. difficile organisms, and hyperimmune anti-C.difficile IgY on the same schedule as Group B. The anti-C. difficile IgYwas prepared as described in Example 1 except that the 12% PEGpreparation was dissolved in one fourth the original yolk volume ofEnsure (Ross Laboratories, Columbus, Ohio).

[0074] All animals were observed for the onset of diarrhea or otherdisease symptoms and death. Each animal was housed in an individual cageand was offered food and water ad libitum. The results are shown inTable 5. TABLE 5 The Effect of Oral Feeding of Hyperimmune IgY Antibodyon C. Difificile Infection TIME TO TIME TO ANIMAL GROUP DIARRHEA^(a)DEATH^(a) A pre-immune IgY only no diarrhea no deaths B Clindamycin, C.difficile, 30 hrs. 49 hrs. preimmune IgY C Clindamycin, C. difficile, 33hrs. 56 hrs. immune IgY

[0075] Hamsters in the control group A did not develop diarrhea andremained healthy during the experimental period. Hamsters in groups Band C developed diarrheal disease. Anti-C. difficile IgY did not protectthe animals from diarrhea or death, all animals succumbed in the sametime interval as the animals treated with preimmune IgY. Thus, whileimmunization with whole organisms apparently can improve sub-lethalsymptoms with particular bacteria (see U.S. Pat. No. 5,080,895 to H.Tokoro), such an approach does not prove to be productive to protectagainst the lethal effects of C. difficile.

Example 3 Production of C. Botulinum Type A Antitoxin in Hens

[0076] In order to determine whether antibodies could be raised againstthe toxin produced by Clostridial pathogens, which would be effective intreating Clostridial diseases, antitoxin to botulinum type A toxin wasproduced. This example involves: (a) toxin modification; (b)immunization; (c) antitoxin collection; (d) antigenicity assessment; and(e) assay of antitoxin titer.

[0077] (a) Toxin Modification. Botulinum type A toxoid was obtained fromB. R. DasGupta. From this, the active type A neurotoxin (M. W.approximately 150 kD) was purified to greater than 99% purity, accordingto published methods. [B. R. DasGupta & V. Sathyamoorthy, Toxicon,22:415 (1984).] The neurotoxin was detoxified with formaldehydeaccording to published methods. [B. R. Singh & B. R. Das Gupta, Toxicon,27:403 (1989).]

[0078] (b) Immunization. Botulinum toxoid for immunization was dissolvedin PBS (1 mg/ml) and was emulsified with an approximately equal volumeof CFA (GIBCO) for initial immunization or IFA for booster immunization.On day zero, two white leghorn hens, obtained from local breeders, wereeach injected at multiple sites (intramuscular and subcutaneous) with 1ml inactivated toxoid emulsified in 1 ml CFA. Subsequent boosterimmunizations were made according to the following schedule for day ofinjection and toxoid amount: days 14 and 21-0.5 mg; day 171-0.75 mg;days 394, 401, 409-0.25 mg. One hen received an additional booster of0.150 mg on day 544.

[0079] (c) Antitoxin Collection. Total yolk immunoglobulin (IgY) wasextracted as described in Example 1(c) and the IgY pellet was dissolvedin the original yolk volume of PBS thimerosal.

[0080] (d) Antigenicity Assessment. Eggs were collected from day 409through day 423 to assess whether the toxoid was sufficientlyimmunogenic to raise antibody Eggs from the two hens were pooled andantibody was collected as described in the standard PEG protocol(Example 1(c)). Antigenicity of the botulinal toxin was assessed onWestern blots. The 150 kD detoxified type A neurotoxin and unmodified,toxic, 300 kD botulinal type A complex (toxin used for intragastricroute administration for animal gut neutralization experiments; seeExample 6) were separated on a SDS-polyacrylamide reducing gel. TheWestern blot technique was performed according to the method of Towbin.[H. Towbin et al., Proc. Nat'l Acad. Sci. USA, 76:4350 (1979).] Ten μgsamples of botulinum complex and toxoid were dissolved in SDS reducingsample buffer (1% SDS, 0.5% 2-mercaptoethanol, 50 mM Tris, pH 6.8, 10%glycerol, 0.025% w/v bromophenol blue, 10% P-mercaptoethanol), heated at95° C. for 10 min and separated on a 1 mm thick 5% SDS-polyacrylamidegel. [K. Weber and M. Osborn, The Proteins, 3d ed., (H. Neurath and R.L. Hill, eds.), pp. 179-223, Academic Press, NY, (1975).] Part of thegel was cut off and the proteins were stained with Coomassie Blue. Theproteins in the remainder of the gel were transferred to nitrocelluloseusing the Milliblot-SDE electro-blotting system (Millipore) according tomanufacturer's directions. The nitrocellulose was temporarily stainedwith 10% Ponceau S [S. B. Carroll and A. Laughon, DNA Cloning: APractical Approach, Vol.III, (D. Glover, ed.), pp. 89-111, IRL Press,Oxford, (1987)] to visualize the lanes, then destained by running agentle stream of distilled water over the blot for several minutes. Thenitrocellulose was immersed in PBS containing 3% BSA overnight at 4° C.to block any remaining protein binding sites.

[0081] The blot was cut into strips and each strip was incubated withthe appropriate primary antibody. The avian botulinum antibodies(described in (c)) and pre-immune chicken antibody (as control) werediluted 1:125 in PBS containing 1 mg/ml BSA for 2 hours at roomtemperature. The blots were washed with two changes each of largevolumes of PBS, BBS-Tween and PBS, successively (10 min/wash). Goatanti-chicken IgG alkaline phosphatase conjugated secondary antibody(Fisher Biotech) was diluted 1:500 in PBS containing 1 mg/ml BSA andincubated with the blot 2 hours at room temperature. The blots werewashed with two changes each of large volumes of PBS and BBS-Tween,followed by one change of PBS and 0.1 M Tris-HCl, pH 9.5. Blots weredeveloped in freshly prepared alkaline phosphatase substrate buffer (100μg/ml NitroBlue Tetrazolium (Sigma), 50 μg/ml 5-bromo-4-chloro-3-indolylphosphate (Sigma), 5 mM MgCl₂ in 50 mM Na₂CO₃, pH 9.5).

[0082] The Western blots are shown in FIG. 1. The antibotulinum IgYreacted to the toxoid to give a broad immunoreactive band at about145-150 kD on the reducing gel.

[0083] This toxoid is refractive to disulfide cleavage by reducingagents due to formalin crosslinking. The immune IgY reacted with theactive toxin complex, a 97 kD botulinum type A heavy chain and a 53 kDlight chain. The preimmune IgY was unreactive to the botulinum complexor toxoid in the Western blot.

[0084] (e) Antitoxin Antibody Titer. The IgY antibody titer to botulinumtype A toxoid of eggs harvested between day 409 and 423 was evaluated byELISA, prepared as follows; Ninety-six-well Falcon Pro-bind plates werecoated overnight at 4° C. with 100 μl/well toxoid (B. R. Singh & B. R.Das Gupta, Toxicon 27:403 (1989)) at 2.5 μg/ml in PBS, pH 7.5 containing0.005% thimerosal. The following day the wells were blocked with PBScontaining 1% BSA for 1 hour at 37° C.

[0085] The IgY from immune or preimmune eggs was diluted in PBScontaining 1% BSA and 0.05% Tween 20 and the plates were incubated for 1hour at 37° C. The plates were washed three times with PBS containing0.05% Tween 20 and three times with PBS alone. Alkalinephosphatase-conjugated goat-anti-chicken IgG (Fisher Biotech) wasdiluted 1:750 in PBS containing 1% BSA and 0.05% Tween 20, added to theplates, and incubated 1 hour at 37° C. The plates were washed as before,and p-nitophenyl phosphate (Sigma) at 1 mg/ml in 0.05 M Na₂CO₃₁ pH 9.5,10 mM MgCl₂ was added.

[0086] The results are shown in FIG. 2. Chickens immunized with thetoxoid generated high titers of antibody to the immunogen. Importantly,eggs from both immunized hens had significant anti-immunogen antibodytiters as compared to preimmune control eggs. The anti-botulinum IgYpossessed significant activity, to a dilution of 1:93,750 or greater.

Example 4 Preparation of Avian Egg Yolk Immunoglobulin in an OrallyAdministrable Form

[0087] In order to administer avian IgY antibodies orally toexperimental mice, an effective delivery formula for the IgY had to bedetermined. The concern was that if the crude IgY were dissolved in PBS,the saline in PBS would dehydrate the mice, which might prove harmfulover the duration of the study. Therefore, alternative methods of oraladministra-tion of IgY were tested. The example involved: (a) isola-tionof immune IgY; (b) solubilization of IgY in water or PBS, includingsubsequent dialysis of the IgY-PBS solution with water to eliminate orreduce the salts (salt and phosphate) in the buffer; and (c) comparisonof the quantity and activity of recovered IgY by absorbance at 280 nmand PAGE, and enzyme-linked immunoassay (ELISA).

[0088] (a) Isolation of Immune IgY. In order to investigate the mosteffective delivery formula for IgY, we used IgY which was raised againstCrotalus durissus terrificus venom. Three eggs were collected from hensimmunized with the C. durissus terrificus venom and IgY was extractedfrom the yolks using the modified Polson procedure described by Thalleyand Carroll [Bio/Technology, 8:934-938 (1990)] as described in Example1(c).

[0089] The egg yolks were separated from the whites, pooled, and blendedwith four volumes of PBS. Powdered PEG 8000 was added to a concentrationof 3.5%. The mixture was centrifuged at 10,000 rpm for 10 minutes topellet the precipitated protein, and the supernatant was filteredthrough cheesecloth to remove the lipid layer. Powdered PEG 8000 wasadded to the supernatant to bring the final PEG concentration to 12%(assuming a PEG concentration of 3.5% in the supernatant). The 12%PEG/IgY mixture was divided into two equal volumes and centrifuged topellet the IgY.

[0090] (b) Solubilization of the IgY in Water or PBS. One pellet wasresuspended in ½ the original yolk volume of PBS, and the other pelletwas resuspended in ½ the original yolk volume of water. The pellets werethen centrifuged to remove any particles or insoluble material. The IgYin PBS solution dissolved readily but the fraction resuspended in waterremained cloudy.

[0091] In order to satisfy anticipated sterility requirements for orallyadministered antibodies, the antibody solution needs to befilter-sterilized (as an alternative to heat sterilization which woulddestroy the antibodies). The preparation of IgY resuspended in water wastoo cloudy to pass through either a 0.2 or 0.45 μm membrane filter, so10 ml of the PBS resuspended fraction was dialyzed overnight at roomtemperature against 250 ml of water. The following morning the dialysischamber was emptied and refilled with 250 ml of fresh H₂O for a seconddialysis. Thereafter, the yields of soluble antibody were determined atOD₂₈₀ and are compared in Table 6. TABLE 6 Dependence of IgY Yield onSolvents ABSORBANCE OF 1:10 PERCENT FRACTION DILUTION AT 280 nm RECOVERYPBS dissolved 1.149 100% H₂O dissolved 0.706  61% PBS dissolved/H₂Odialyzed 0.885  77%

[0092] Resuspending the pellets in PBS followed by dialysis againstwater recovered more antibody than directly resuspending the pellets inwater (77% versus 61%). Equivalent volumes of the IgY preparation in PBSor water were compared by PAGE, and these results were in accordancewith the absorbance values (data not shown).

[0093] (c) Activity of IgY Prepared with Different Solvents. An ELISAwas performed to compare the binding activity of the IgY extracted byeach procedure described above. C. durissus terrificus (C.d.t.) venom at2.5 μg/ml in PBS was used to coat each well of a 96-well microtiterplate. The remaining protein binding sites were blocked with PBScontaining 5 mg/ml BSA. Primary antibody dilutions (in PBS containing 1mg/ml BSA) were added in duplicate. After 2 hours of incubation at roomtemperature, the unbound primary antibodies were removed by washing thewells with PBS, BBS-tween, and PBS. The species specific secondaryantibody (goat anti-Chicken immunoglobulin alkaline-phosphataseconjugate; Sigma Chemical Co.) was diluted 1:750 in PBS containing 1mg/ml BSA and added to each well of the microtiter plate. After 2 hoursof incubation at room temperature, the unbound secondary antibody wasremoved by washing the plate as before, and freshly prepared alkalinephosphatase substrate (Sigma Chemical Co.) at 1 mg/ml in 50 mM Na₂CO₃,10 mM MgCl₂, pH 9.5 was added to each well. The color development wasmeasured on a Dynatech MR 700 microplate reader using a 412 nm filter.The results are shown in Table 7. TABLE 7 Antigen-Binding Activity ofIgY Prepared with Different Solvents PBS H₂O PBS/ DILUTION PREIMMUNEDISSOLVED DISSOLVED H₂O 1:500 0.005 1.748 1.577 1.742 1:2,500 0.0040.644 0.349 0.606 1:12,500 0.001 0.144 0.054 0.090 1:62,500 0.001 0.0250.007 0.016 1:312,500 0.010 0.000 0.000 0.002

[0094] The binding assay results parallel the recovery values in Table6, with PBS-dissolved IgY showing slightly more activity than thePBS-dissolved/H₂O dialyzed antibody. The water-dissolved antibody hadconsiderably less binding activity than the other preparations.

Example 5 Survival of Antibody Activity After Passage Through theGastrointestinal Tract

[0095] In order to determine the feasibility of oral administration ofantibody, it was of interest to determine whether orally administeredIgY survived passage through the gastrointestinal tract. The exampleinvolved: (a) oral administration of specific immune antibody mixed witha nutritional formula; and (b) assay of antibody activity extracted fromfeces.

[0096] (a) Oral Administration of Antibody. The IgY preparations used inthis example are the same PBS-dissolved/H₂O dialyzed antivenom materialsobtained in Example 4 above, mixed with an equal volume of ENFAMIL (RossLaboratories). Two mice were used in this experiment, each receiving adifferent diet as follows:

[0097] 1) water and food as usual;

[0098] 2) immune IgY prep dialyzed against water and mixed 1:1 withENFAMIL. (The mouse was given this mixture as its only source of foodand water).

[0099] (b) Antibody Activity After Ingestion. After both mice hadingested their respective fluids, each tube was refilled withapproximately 10 ml of the appropriate fluid first thing in the morning.By mid-morning there was about 4 to 5 ml of liquid left in each tube. Atthis point stool samples were collected from each mouse, weighed, anddissolved in approximately 500 μl PBS per 100 mg stool sample. Onehundred and sixty mg of control stools (no antibody) and 99 mg ofexperimental stools (specific antibody) in 1.5 ml microfuge tubes weredissolved in 800 and 500 μl PBS, respectively. The samples were heatedat 37° C. for 10 minutes and vortexed vigorously. The experimentalstools were also broken up with a narrow spatula. Each sample wascentrifuged for 5 minutes in a microfuge and the supernatants,presumably containing the antibody extracts, were collected. The pelletswere saved at 2-8° C. in case future extracts were needed. Because thesupernatants were tinted, they were diluted five-fold in PBS containing1 mg/ml BSA for the initial dilution in the enzyme immunoassay (ELISA).The primary extracts were then diluted five-fold serially from thisinitial dilution. The volume of primary extract added to each well was190 μl. The ELISA was performed exactly as described in Example 4. TABLE8 Specific Antibody Activity After Passage Through the GastrointestinalTract CONTROL FECAL EXP. FECAL DILUTION PREIMMUNE IgY EXTRACT EXTRACT1:5 <0 0.000 0.032 1:25 0.016 <0 0.016 1:125 <0 <0 0.009 1:625 <0 0.0030.001 1:3125 <0 <0 0.000

[0100] There was some active antibody in the fecal extract from themouse given the specific antibody in ENFAMIL formula, but it was presentat a very low level. Since the samples were assayed at an initial 1:5dilution, the binding observed could have been higher with less dilutesamples. Consequently, the mice were allowed to continue ingestingeither regular food and water or the specific IgY in ENFAMIL formula, asappropriate, so the assay could be repeated. Another ELISA plate wascoated overnight with 5 μg/ml of C.d.t. venom in PBS.

[0101] The following morning the ELISA plate was blocked with 5 mg/mlBSA, and the fecal samples were extracted as before, except that insteadof heating the extracts at 37° C., the samples were kept on ice to limitproteolysis. The samples were assayed undiluted initially, and in 5×serial dilutions thereafter. Otherwise the assay was carried out asbefore. TABLE 9 Specific Antibody Survives Passage Through theGastrointestinal Tract CONTROL DILUTION PREIMMUNE IgY EXTRACT EXP.EXTRACT undiluted 0.003 <0 0.379 1:5 <0 <0 0.071 1:25 0.000 <0 0.0271:125 0.003 <0 0.017 1:625 0.000 <0 0.008 1:3125 0.002 <0 0.002

[0102] The experiment confirmed the previous results, with the antibodyactivity markedly higher. The control fecal extract showed noanti-C.d.t. activity, even undiluted, while the fecal extract from theanti-C.d.t. IgY/ENFAMIL-fed mouse showed considerable anti-C.d.t.activity. This experiment (and the previous experiment) clearlydemonstrate that active IgY antibody survives passage through the mousedigestive tract, a finding with favorable implications for the successof IgY antibodies administered orally as a therapeutic or prophylactic.

Example 6 In Vivo Neutralization of Type A Botulinum Neurotoxin by AvianAntitoxin Antibody

[0103] This example demonstrated the ability of PEG-purified antitoxin,collected as described in Example 3, to neutralize the lethal effect ofbotulism neurotoxin type A in mice. To determine the oral lethal dose(LD₁₀₀) of Botulism toxin A, groups of BALB/C mice were given differentdoses of toxin per unit body weight (average body weight of 24 grams).For oral administration, toxin A complex, which contains the neurotoxinassociated with other non-toxin proteins was used. This complex ismarkedly more toxic than purified neurotoxin when given by the oralroute. [I. Ohishi et al., Infect. and Immun., 16:106 (1977).] Botulismtoxin type A complex, obtained from Eric Johnson (University OfWisconsin, Madison) was 250 μg/ml in 50 mM sodium citrate, pH 5.5,specific toxicity 3×10⁷ mouse LD₅₀/mg with parenteral administration.Approximately 40-50 ng/gm body weight was usually fatal within 48 hoursin mice maintained on conventional food and water. When mice were givena diet ad libitum of only Enfamil (Mead Johnson), the concentrationneeded to produce lethality was approximately 2.5 times higher (125ng/gm body weight). Botulism concentrations of approximately 200 ng/gmbody weight was fatal in mice fed Enfamil containing preimmune IgY(resuspended in Enfamil at the original yolk volume).

[0104] The oral LD₁₀₀ of botulinum toxin was also determined in micethat received known amounts of a mixture of preimmune IgY-Ensure (RossLaboratories) delivered orally through feeding needles. Using a 22 gaugefeeding needle, mice were given 250 μl each of a preimmune IgY-Ensuremixture (preimmune IgY dissolved in ¼ original yolk volume) 1 hourbefore and ½ hour and 5 hours after administering botulinal toxin. Toxinconcentrations given orally ranged from approximately 12 to 312 ng/gmbody weight (0.3 to 7.5 μg per mouse. Botulism complex concentration ofapproximately 40 ng/gm body weight (1 μg per mouse) was lethal in allmice in less than 36 hours.

[0105] Two groups of BALB/c mice, 10 per group, were each given orally asingle dose of 1 μg each of botulinal toxin complex in 100 μl of 50 mMsodium citrate pH 5.5. The mice received 250 μl treatments of a mixtureof either preimmune or immune IgY in Ensure (¼ original yolk volume) 1hour before and ½ hour, 4 hours, and 8 hours after botulinal toxinadministration. The mice received three treatments per day for two moredays. The mice were observed for 96 hours. The survival and mortalityare shown in Table 10. TABLE 10 Neutralization of Botulinal Toxin A InVivo TOXIN DOSE NUMBER OF NUMBER OF ng/gm ANTIBODY TYPE MICE ALIVE MICEDEAD 41.6 non-immune  0 10 41.6 anti- 10  0 botulinal toxin

[0106] All mice treated with the preimmune IgY-Ensure mixture diedwithin 46 hours post-toxin administration. The average time of death inthe mice was 32 hours post toxin administration. Treatments of preimmuneIgY-Ensure mixture did not continue beyond 24 hours due to extensiveparalysis of the mouth in mice of this group. In contrast, all ten micetreated with the immune anti-botulinal toxin IgY-Ensure mixture survivedpast 96 hours. Only 4 mice in this group exhibited symptoms of botulismtoxicity (two mice about 2 days after and two mice 4 days after toxinadministration).

[0107] These mice eventually died 5 and 6 days later. Six of the mice inthis immune group displayed no adverse effects to the toxin and remainedalive and healthy long term. Thus, the avian anti-botulinal toxinantibody demonstrated very good protection from the lethal effects ofthe toxin in the experimental mice.

Example 7 Production of an Avian Antitoxin Against Clostridium DifficileToxin A

[0108] Toxin A is a potent cytotoxin secreted by pathogenic strains ofC. difficile that plays a direct role in damaging gastrointestinaltissues. In more severe cases of C. difficile intoxication,pseudomembranous colitis can develop which may be fatal. This would beprevented by neutralizing the effects of this toxin in thegastrointestinal tract. As a first step, antibodies were producedagainst a portion of the toxin. The example involved: (a) conjugation ofa synthetic peptide of toxin A to bovine serum albumin; (b) immunizationof hens with the peptide-BSA conjugate; and (c) detection of antitoxinpeptide antibodies by ELISA.

[0109] (a) Conjugation of a Synthetic Peptide of Toxin A to Bovine SerumAlbumin. The synthetic peptide CQTIDGKKYYFN-NH₂ was preparedcommercially (Multiple Peptide Systems, San 25. Diego, Calif.) andvalidated to be >80% pure by high-pressure liquid chromatography. Theeleven amino acids following the cysteine residue represent a consensussequence of a repeated amino acid sequence found in Toxin A [Wren etal., Infection and Immunity, 59:3151-3155 (1991)]. The cysteine wasadded to facilitate conjugation to carrier protein.

[0110] In order to prepare the carrier for conjugation. BSA (Sigma) wasdissolved in 0.01 M NaPO₄, pH 7.0 to a final concentration of 20 mg/mland n-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS; Pierce) wasdissolved in N,N-dimethyl formamide to a concentration of 5 mg/ml. MBSsolution, 0.51 ml, was added to 3.25 ml of the BSA solution andincubated for 30 minutes at room temperature with stirring every 5minutes. The MBS-activated BSA was then purified by chromatography on aBio-Gel P-10 column (Bio-Rad; 40 ml bed volume) equilibrated with 50 mMNaPO₄, pH 7.0 buffer. Peak fractions were pooled (6.0 ml).

[0111] Lyophilized toxin A peptide (20 mg) was added to the activatedBSA mixture, stirred until the peptide dissolved and incubated 3 hoursat room temperature. Within 20 minutes, the reaction mixture becamecloudy and precipitates formed. After 3 hours, the reaction mixture wascentrifuged at 10,000×g for 10 min and the supernatant analyzed forprotein content. No significant protein could be detected at 280 nm. Theconjugate precipitate was washed three times with PBS and stored at 4°C. A second conjugation was performed with 15 mg of activated BSA and 5mg of peptide and the conjugates pooled and suspended at a peptideconcentration of 10 mg/ml in 10 mM NaPO₄, pH 7.2.

[0112] (b) Immunization of Hens with Peptide Conjugate. Two hens wereeach initially immunized on day zero by injection into two subcutaneousand two intramuscular sites with 1 mg of peptide conjugate that wasemulsified in CFA (GIBCO). The hens were boosted on day 14 and day 21with 1 mg of peptide conjugate emulsified in IFA (GIBCO).

[0113] (c) Detection of Antitoxin Peptide Antibodies by ELISA. IgY waspurified from two eggs obtained before immunization (pre-immune) and twoeggs obtained 31 and 32 days after the initial immunization using PEGfractionation as described in Example 1.

[0114] Wells of a 96-well microtiter plate (Falcon Pro-Bind Assay Plate)were coated overnight at 4° C. with 100 μg/ml solution of the toxin Asynthetic peptide in PBS, pH 7.2 prepared by dissolving 1 mg of thepeptide in 1.0 ml of H₂O and dilution of PBS. The pre-immune and immuneIgY preparations were diluted in a five-fold series in a buffercontaining 1% PEG 8000 and 0.1% tween-20 (v/v) in PBS, pH 7.2. The wellswere blocked for 2 hours at room temperature with 150 μl of a solutioncontaining 5% (v/v) Carnation nonfat dry milk and 1% PEG 8000 in PBS, pH7.2. After incubation for 2 hours at room temperature, the wells werewashed, secondary rabbit anti-chicken IgG-alkaline phosphatase (1:750)added, the wells washed again and the color development obtained asdescribed in Example 1. The results are shown in Table 11. TABLE 11Reactivity of IgY with Toxin Peptide ABSORBANCE AT 410 nm DILUTION OFPEG IMMUNE ANTI- PREP PREIMMUNE PEPTIDE 1:100 0.013 0.253 1:500 0.0040.039 1:2500 0.004 0.005

[0115] Clearly, the immune antibodies contain titers against thisrepeated epitope of toxin A.

[0116] From the above it should be clear that the present inventionprovides compositions and methods for effective therapy againstclostridial toxin disease therapy. The antitoxins can also be used fordiagnostic use.

What is claimed is:
 1. A method, comprising: a) providing: i) a subject;and ii) an orally administrable aqueous solution comprising avianantitoxin; and b) orally administering said avian antitoxin to saidsubject.
 2. The method of claim 1, wherein said antitoxin comprisesbotulinus antitoxin.
 3. The method of claim 1, wherein said aqueoussolution comprises a nutritional formula.
 4. The method of claim 3,wherein said nutritional formula comprises infant formula.